Cathepsin B Is Involved in the Trafficking of TNF- Α-Containing Vesicles to the Plasma Membrane in Macrophages

Cathepsin B Is Involved in the Trafficking of TNF- α-Containing Vesicles to the Plasma Membrane in Macrophages

This information is current as Soon-Duck Ha, Andrew Martins, Khashayarsha Khazaie, of September 27, 2021. Jiahuai Han, Bosco M. C. Chan and Sung Ouk Kim J Immunol 2008; 181:690-697; ; doi: 10.4049/jimmunol.181.1.690 http://www.jimmunol.org/content/181/1/690 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cathepsin B Is Involved in the Trafﬁcking of TNF-␣-Containing Vesicles to the Plasma Membrane in Macrophages1

Soon-Duck Ha,*† Andrew Martins,*† Khashayarsha Khazaie,‡ Jiahuai Han,§ Bosco M. C. Chan,* and Sung Ouk Kim2*†

TNF-␣ is a potent proinflammatory cytokine, essential for initiating innate immune responses against invading microbes and a key mediator involved in the pathogenesis of acute and chronic inflammatory diseases. To identify molecules involved in the production of TNF-␣, we used a functional gene identification method using retroviral integration-mediated mutagenesis, followed by LPS-stimulated TNF-␣ production analysis in macrophages. We found that cathepsin B, a lysosomal cysteine proteinase, was required for optimal posttranslational processing of TNF-␣ in response to the bacterial cell wall component LPS. Mouse bone Downloaded from marrow-derived macrophages from cathepsin B-deficient mice and macrophages treated with the cathepsin B-specific chemical inhibitor CA074 methyl ester or small interfering RNA against cathepsin B secreted significantly less TNF-␣ than wild-type or nontreated macrophages. We further showed that the inhibition of cathepsin B caused accumulation of 26-kDa pro-TNF-containing vesicles. Ectopic expression of GFP-conjugated pro-TNF further suggests that pro-TNF failed to reach the plasma membrane without intracellular cathepsin B activity. Altogether, these data suggest that intracellular cathepsin B activity is involved in the TNF-␣-containing vesicle trafficking to the plasma membrane. The Journal of Immunology, 2008, 181: 690–697. http://www.jimmunol.org/

roduction of the potent inflammatory cytokine TNF-␣ in cellular Ag-1 that promotes mRNA degradation or inhibits protein response to invading microbes is a crucial step for mount- translation, respectively (6). P ing initial innate immune responses. However, uncon- TNF-␣ is encoded as a 26-kDa type II transmembrane precursor trolled production of TNF-␣ is linked to the pathogenesis of severe (pro-TNF), which is transported from the trans-Golgi network to acute and chronic inflammatory diseases (1). Thus, the biosynthe- the recycling endosome (7), then delivered to the cell surface sis and release of TNF-␣ should be tightly regulated at different where pro-TNF undergoes proteolytic cleavage by the TNF-␣-con- levels to prevent inadvertent production under normal condition verting enzyme (8–11). Membrane fusion between the TNF-␣- (1–4). Activation of macrophages by the bacterial cell wall com- containing vesicles from trans-Golgi network and the recycling by guest on September 27, 2021 ponent LPS is mediated through TLR4, resulting in the recruitment endosomes, and between the recycling endosomes and the cell of signaling adaptors such as MyD88 and TRIF (5). This recruit- surface membrane was shown to be mediated through interactions ment of signaling adaptors to TLR4 induces activation of signaling between various trans-SNARE (soluble-N-ethylmaleimide-sensi- cascades involving multiple signaling molecules, including the tive factor-attachment protein (SNAP)3 receptor) family members family of protein serine kinases IL-1R-associated kinase 1 and (4). Recent studies have shown that TNF-␣ surface delivery and kinase 4 and the adaptor molecule TNFR-associated factor 6. Sub- secretion is regulated through a process involving cholesterol-de- sequent activation of MAPKs and transcription factor NF-␬B oc- pendent lipid raft formation at the phagocytic cup of activated curs, which controls transcription of cytokines including TNF-␣ macrophages (7, 12); however, it is unknown how TNF-␣ surface (5). Additional layers of TNF-␣ production control occur at the delivery is regulated and whether it can be facilitated upon TLR level of translation. TNF-␣ mRNA contains an AU-rich element in activation. We used a functional gene identification procedure us- its 3Ј untranslated region, which determines its half-life and trans- ing retrovirus integration-generated mutagenesis (13–15) and identi- lational efficiency through binding tristetraprolin or T cell intra- fied that cathepsin B activity was required for optimal TNF-␣ trans- portation to the plasma membrane. Cathepsin B is a lysosomal cysteine protease involved in the degradation of cellular proteins in lysosomes. Unlike other cathepsins, cathepsin B functions as an en- *Department of Microbiology and Immunology, and †Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, dopeptidase at neutral pH and is also found in extralysosomal sites, Ontario, Canada; ‡Department of Microbiology-Immunology, Northwestern Univer- including the cytosol, the plasma membrane, and pericellular spaces, sity, Chicago, IL 60611-3015; and §The Scripps Research Institute, La Jolla, CA where it participates in various cellular processes including cancer 92037 metastasis, inflammation, myoblast differentiation, IL-1␤ production, Received for publication February 27, 2008. Accepted for publication April 27, 2008. and cell death (16–21). In this study, we demonstrate that cathepsin B The costs of publication of this article were defrayed in part by the payment of page is involved in the posttranslational process of TNF-␣, likely in the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. trafficking of TNF-␣-containing vesicles to the plasma membrane. 1 This work was supported by Research Grant MOP68841 from the Canadian Institute of Health (to S.O.K.) and by National Institutes of Health Research Grant R01- CA104547-01 (to K.K.). 3 Abbreviations used in this paper: SNAP, soluble-N-ethylmaleimide-sensitive factor- 2 Address correspondence and reprint requests to Dr. Sung Ouk Kim, Infectious Dis- attachment protein; SNARE, SNAP receptor; siRNA, small interfering RNA; eases Research Group, Siebens-Drake Research Institute, Room 119, University of BMDIM, bone marrow-derived immortalized macrophage. Western Ontario, 1400 Western Road, London, Ontario N6G 2V4, Canada. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 691

Materials and Methods room temperature Nucleofector solution V to a final concentration of 2.0 ϫ 6 ␮ Materials and reagents 10 cells/100 l, and mixed with siRNA (Dharmacon), for human cathepsin B (NM_001908, ON-TARGET Plus Duplex J-004266-13-0005). Cathepsin B inhibitor III, cathepsin B inhibitor IV, cathepsin K inhibitor Transfection was performed using the Nucleofector I device (Amaxa Bio- III, cathepsin G inhibitor I, and calpain inhibitor III (zVF-Cho) were pur- systems). At 48 h after nucleofection, cells were plated and stimulated with chased from Calbiochem (EMD Bioscience). Cathepsin B and cathepsin L LPS for TNF-␣ assay. Other cells were harvested for cathepsin B protein inhibitor (zFF-fmk) was purchased from Sigma-Aldrich. Abs for p38 knockdown analysis. The verification of cathepsin B knockdown was per- MAPK and cathepsin B were obtained from Cell Signaling Technology formed by Western blot analysis using anti-human cathepsin B Ab and Calbiochem, respectively. Mouse and human TNF-␣ Abs were pur- (eBioscience). chased from eBioscience. Total cell lysate preparation and immunoblotting Cell culture Total cell lysate preparation and immunoblotting procedures were per- Mouse bone marrow-derived immortalized macrophages (BMDIM) were formed as previously described (25). Briefly, cells were lysed in ice-cold prepared as previously described (22–24) from C57BL/6 mice. Bone mar- lysis buffer (20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 1 mM Na3VO4, row-derived macrophages from cathepsin B gene (Ctsb)-deficient 40 mM ␤-glycerophosphate, 30 mM NaF, and 20 mM sodium pyrophos- (CtsbϪ/Ϫ) or control strain C57BL/6 (Ctsbϩ/ϩ) mice or from the human phate (pH 7.2)) containing a protease inhibitor cocktail (Roche). The cell monocytic cell line THP-1 were cultured in RPMI 1640 medium contain- lysates were incubated on ice for 10 min and centrifuged at 12,500 rpm for ing 10% heat-inactivated FBS (Sigma-Aldrich), 10 mM MEM nonessential 15 min at 4°C. The supernatants were electrophoretically resolved in SDS- amino acid solution, 100 U/ml penicillin G sodium, 100 ␮g/ml streptomy- polyacrylamide gels, followed by transfer onto nitrocellulose membranes cin sulfate, and 1 mM sodium pyruvate. Cells were grown at 37°C in the (Bio-Rad). The membranes were blocked at room temperature for 1 h with 5% (w/v) skim milk, and then incubated overnight at room temperature humidified atmosphere of 5% CO2. Human PBMC were isolated from with the primary Ab. The membranes were washed and developed using an whole blood as previously described (25). Downloaded from ECL detection system (Pierce). Mutagenesis with retrovirus, screening for defects in TNF-␣ Metabolic radiolabeling and immunoprecipitation production, and identification of retroviral targeting gene BMDIM were plated 2.5 ϫ 106 cell/well. Cells pretreated with or without BMDIM were transfected with the pDisrup8-loxP retroviral vector con- the cathepsin B inhibitor CA074 methyl ester (CA-Me) for 45 min were taining a blasticidin resistance gene as previously described (15), except a stimulated with LPS 1 ␮g/ml for 90 min. LPS-stimulated cells, or untreated loxP sequence was incorporated into the vector. Each blasticidin resistance cells as the control, were replaced with methionine-free medium for 60 min clone was cultured for 2 wk and tested for TNF-␣ production in response http://www.jimmunol.org/ under the LPS (1 ␮g/ml). They were then labeled for 1 h with 200 ␮Ci/ml to LPS (1 ␮g/ml). TNF-␣ bioassay was performed as previously described of [35S]methionine (PE Life and Analytical Science). The labeled cells (26). To rescue retroviral insertion-induced mutations, CTSBmut, a partial were washed and replaced with fresh regular growth medium. At the time mRNA sequence of the fused gene product, was transfected with a retro- interval indicated, medium and cells were collected. Cells were lysed viral vector expressing Cre (pBaBe-Cre) and puromycin resistance gene. using ice-cold lysis buffer (20 mM MOPS, 2 mM EGTA, 5 mM EDTA, Cre-expressing cells were then selected by culturing cells in the presence 1mMNaVO ,40mM␤-glycerophosphate, 30 mM NaF, and 20 mM of puromycin (10 ␮g/ml). All puromycin resistance cells became sensitive 3 4 sodium pyrophosphate (pH 7.2)) containing protease inhibitor cocktails to blasticidin. Identification of the gene targeted by pDisrup8pLox was (Roche). TNF-␣ was immunoprecipitated from the samples, using anti- performed using 3Ј-RACE as previously described (15). Primers used for mouse TNF-␣ Ab (eBioscience), separated on SDS-polyacrylamide gels semiquantitative analysis for pro-retrovirus, cathepsin B, and GAPDH are and visualized by autoradiography. as follow: pro-retrovirus (BSR4) 5Ј-GTGAAGGACAGTGATGGAC by guest on September 27, 2021 AG-3Ј and cathepsin B (antisense) 5Ј-GCCCTAAGGACTGGACAATGA- Flow cytometry analysis 3Ј; cathepsin B (sense) 5Ј-TGCAGGCCCAGGCTGTCG-3Ј and (antisense) 5Ј-GCCCTAAGGACTGGACAATGA-3Ј; and GAPDH (sense) 5Ј- BMDIM were treated with or without cathepsin B inhibitors for 30 min GCATTGTGGAAGGGCTCATG-3Ј and (antisense) 5Ј-TTGCTGTTG before stimulation with LPS (100 ng/ml) for 18 h. Cells were then fixed in AAGTCGCAGGAG-3Ј. 1% formalin for 10 min with periodic agitation. After two washes with PBS, cells were then resuspended and incubated for 30 min on ice in PBS Measurement of cathepsin B activity containing 0.1% saponin and 10% FBS. TNF-␣ was then stained using a PE-labeled anti-mouse TNF-␣ Ab (eBioscience) for 30 min. After two final Cathepsin B activity was measured by cathepsin B activity assay kit (Bio- washes, cells were resuspended to 1 million cells/ml in PBS and analyzed Vision). Briefly, cells were washed with the PBS, and lysed in chilled by flow cytometry using a FACSCalibur (BD Biosciences). Data analysis cathepsin B cell lysis buffer. After incubation cell on ice for 10 min, 10 mM was performed using CellQuest software (BD Biosciences). cathepsin B substrate (Ac-RR-AFC, 200 ␮M final concentration) was added and incubated at 37°C for indicated time. The release of free amino- Immunofluorescence staining and TNF-␣ trafficking analysis 4-trifluoromethyl coumarin was monitored in a fluorometer (Fluoroskan ascent FL; Thermo LabSystems) with a 409 nm excitation filter and 515 BMDIM were electroporated with the plasmid endcoding EGFP-TNF (ob- nm emission filter. tained from Dr. J. Stow (University of Queensland, Brisbane, Australia). Transfected cells were fixed with 4% paraformaldehyde and observed Quantitative real-time PCR through Bio-Rad Radiance 2000 Two-Photon fluorescence confocal microscopy. Images were obtained using LaserSharp 2000 software. The ␣ mRNA expression of mouse TNF- in macrophages was quantified on the fixed macrophages were also stained with 10 ␮g/ml Hoechst 33258 Rotor-Gene RG3000 quantitative multiplex PCR instrument using the Bril- (Sigma-Aldrich) in PBS for 2 min to visualize nuclear staining and fol- liant SYBR Green PCR Master mix (Applied Biosystems). Total Cellular lowed by image analysis under confocal microscopy. For immunofluores- RNA was isolated using TRIzol (Life Technologies) according to the man- cence staining, macrophages were permeabilized with 0.1% Triton X-100 ␮ ufacturer’s instructions. Briefly, 4 g of total RNA was reverse transcribed and immunostained for TNF-␣. Endogenous TNF-␣ was detected by im- by using oligo(dT) primers and the Superscript II reverse transcriptase munofluorescence using biotinylated anti-rabbit IgG and fluorescein avidin system (Invitrogen) according to the manufacturer’s recommendations. D (Vector Laboratories), using a Qimaging (Burnaby) cooled charged- ␣ Ј Oligonucleotide primers used were the following: for mouse TNF- ,5- coupled device camera on an Axioscope 2 (Carl Zeiss) microscope. CTGGAAATAGCTCCCAGAA-3Ј (5Ј primer) and 5Ј-CATTTGGGAACT TCTCATCC-3Ј (3Ј primer); and for GAPDH, 5Ј-GCATTGTGGAAGGGC TCATG-3Ј (5Ј primer) and 5Ј-TTGCTGTTGAAGTCGCAGGAG-3Ј (3Ј Results primer). Disruption of Ctsb in macrophages causes a defect in TNF-␣ production Small interfering RNA (siRNA) To identify genes involved in the production of TNF-␣ in response siRNA oligonucleotides directed against human cathepsin B were pur- to LPS, we used retrovirus-mediated mutagenesis in BMDIM. The chased from Dharmacon. Transfection of THP-1 cell with siRNAs was performed using the Nucleofector II kit (Amaxa Biosystems), according to retroviral vector was constructed as previously described (15), the manufacturer’s instructions. Briefly, cells were subcultured 1 day be- except that the loxP site was incorporated at the 3Ј long terminal fore nucleofection. The next day, cells were harvested, resuspended in repeat, which is duplicated during chromosomal insertion (Fig. 1A). 692 CATHEPSIN B IN TNF-␣-CONTAINING VESICLE TRAFFICKING Downloaded from

FIGURE 1. Disruption of Ctsb in macrophages confers defect in TNF-␣ production. A, Diagram of the retroviral vector pDisrup8-loxP used for Ctsb disruption. Rz, Ribozyme sequence; SD, splicing donor; SA, splicing acceptor; pA, poly(A) signal sequence; CMV, CMV promoter; Blasticidinϩ, blasticidin-resistant gene; LTR, long terminal repeat. B, Identiﬁcation of retroviral vector targeting gene. Retroviral vector was inserted in between exon 1 and exon 2 of Ctsb by 3Ј-RACE and DNA sequencing as described in Materials and Methods. C, Generation of null allele of cathepsin B by retroviral

vector and its rescue by pCre. Semiquantitative RT-PCR was performed (left) using a primer targeting the retroviral vector and a cathepsin B-specific primer http://www.jimmunol.org/ (upper panel) or both primers targeting the Ctsb (middle panel). GAPDH was amplified as a control using mouse GAPDH-specific primers (lower panel). CTSBmut, a clone containing cathepsin B-null allele generated by retroviral vector, and CTSBmut-Cre, a clone cured cathepsin B-null allele by Cre macrophages were treated. Western blot analysis (right) was performed in total cell lysates from wild-type, CTSBmut, or CTSBmut-Cre macrophages for cathepsin B and p38 MAPK (for loading control). D, Cathepsin B activity in wild-type, CTSBmut, and CTSBmut-Cre macrophages was measured in total cell lysates as described in Materials and Methods. E, Similarly, these cells were treated with LPS (100 ng/ml) for 6 h and TNF-␣ in the cell culture medium was measured by TNF-␣ bioassay. Data are expressed as mean Ϯ SD (n ϭ 3 experiments). by guest on September 27, 2021 The proviral sequences can readily be excised out from chromo- Cathepsin B activity is involved in the posttranslational some by ectopically expressing Cre recombinase. This feature processing of TNF-␣ in macrophages greatly enhances the convenience in validating the effects of viral To further verify the involvement of cathepsin B in TNF-␣ pro- insertion because excision of provirus should cure the defect induction, bone marrow-derived macrophages from wild-type curred by a viral integration. As described in Materials and Meth- (C57BL/6) and cathepsin B deficient (C57BL/6ctsbϪ/Ϫ) mice were ϳ ods, we screened 1000 blasticidin-resistant retrovirus-mutated treated with various times and doses of LPS, and the levels of ␣ Ϫ Ϫ clones and identified a clone defective in producing TNF- in TNF-␣ production were analyzed. Ctsb / macrophages produced Ј response to LPS. By 3 -RACE of the mRNA fused with blastici- significantly lower levels of TNF-␣ in response to LPS than sim- din, the disrupted gene was identified as Ctsb. A partial mRNA ilarly treated wild-type macrophages (Ctsbϩ/ϩ) (Fig. 2A), even sequence of the fused gene product generated by retroviral inser- though the level of TNF-␣ mRNA was increased in the same ex- mut tion in the clone (termed CTSB ) is shown in Fig. 1B. The blas- tent by LPS (Fig. 2B, bottom). Consistent with these results, there ticidin gene encoded by the viral vector appeared to be inserted in was no difference in the induction of mRNA levels in wild-type, the intron between exon 1 and exon 2 and, therefore, disrupted CTSBmut, and CTSBmut-Cre cells (Fig. 2B, top). To further exam- Ctsb at one allele. Expression of Cre using a retroviral vector con- ine whether the defect in TNF-␣ production in cathepsin B mutant taining the puromycin resistance gene in CTSBmut rendered the macrophages is TLR4-specific or if it applies to stimulation of cells puromycin-resistant but sensitive to blasticidin (data not other TLRs, we treated wild-type, CTSBmut, and CTSBmut-Cre shown), and the Ctsb-fused blasticidin gene mRNA was no longer cells with TLR agonists lipoteichoic acid for TLR2 and CpG for expressed in the Cre-expressing CTSBmut clone (CTSBmut-Cre) TLR9. In all treatments, the levels of TNF-␣ production in (Fig. 1C, left top panel). Based on semiquantitive RT-PCR anal- CTSBmut were significantly lower than production levels found ysis, the level of cathepsin B mRNA was diminished in CTSBmut, in either wild-type or CTSBmut-Cre cells (Fig. 2C). and wild-type mRNA levels were restored by expressing Cre (CTSBmut-Cre) Fig. 1C, left middle panel). Similarly, the level of TNF-␣ production in human monocytes is more sensitive cathepsin B protein in CTSBmut cells decreased to ϳ50% of the to CA-Me than are murine macrophages level found in wild-type or CTSBmut-Cre cells (Fig. 1C, right). We We examined the involvement of cathepsin B in human monocytic further confirmed that total cathepsin B activity and TNF-␣ pro- cell line THP-1 using siRNAs against cathepsin B. THP-1 cells duction induced by LPS in CTSBmut were also reduced to ϳ50% treated with the siRNA against cathepsin B down-regulated the of wild-type cells, and returned to normal levels after excising viral expression of cathepsin B ϳ50% of scrambled siRNA-treated insertion by CRE (CTSBmut-Cre) (Fig. 1D). These results indicate (control) cells. The production of TNF-␣ in response to LPS (100 that the decrease in TNF-␣ production and cathepsin B activity in ng/ml) was diminished to a similar extent (ϳ50%) by treatments CTSBmut were due to a retrovirus-mediated gene disruption. using siRNAs against cathepsin B (Fig. 3A). Because CtsbϪ/Ϫ The Journal of Immunology 693

FIGURE 2. Macrophages deficient in cathepsin B show defects in TNF-␣ production in response to inflammatory stimuli. A, Bone marrow-derived macrophages from wild-type C57BL/6 (Ctsbϩ/ϩ)orCtsbϪ/Ϫ mice were stimulated with LPS (1 ␮g/ml) and production of TNF-␣ was measured in culture medium after the time indicated in the experiment (top). Similarly, production of TNF-␣ was measured after treating cells with various doses of LPS as indicated in the experiment for3h(bottom). B, Macrophages wild-type or deficient in cathepsin B were treated with LPS (1 ␮g/ml) for 3 h and TNF-␣ mut mut mRNA levels were analyzed by real-time RT-PCR. C, Wild-type, CTSB , or CTSB -Cre cells were treated with LPS (100 ng/ml), lipoteichoic acid Downloaded from (LTA; 10 ␮g/ml) or CpG (20 ␮g/ml) for 5 h and TNF-␣ production in culture medium was measured by TNF-␣ bioassay. Data are expressed as mean Ϯ SD (n ϭ 3 experiments). macrophages showed significant but partial defects in TNF-␣ pro- production as CA-Me, but other cathepsin or calpain inhibitors duction, we used the membrane permeable cathepsin B-specific failed to inhibit TNF-␣ production in either wild-type or CtsbϪ/Ϫ inhibitor CA-Me to compare the extent of cathepsin B dependence macrophages (Fig. 3B, right). In human PBMC, CA-Me at 50 ␮M http://www.jimmunol.org/ in TNF-␣ production between mouse bone marrow-derived and inhibited TNF-␣ secretion to ϳ80% similar to the level of inhibi- human THP-1 macrophages. CA-Me inhibited TNF-␣ production tion elicited by a protein transport inhibitor brefeldin A (Fig. 3C). in a dose-dependent manner, resulting in ϳ50% inhibition of TNF-␣ secretion at 5 ␮M, ϳ80% at 50 ␮M, and Ͼ95% at 100 ␮M Cathepsin B activity is required for the posttranslational ␣ in THP-1 cells (Fig. 3B, left). In comparison, mouse bone marrow- processing of TNF- derived macrophages were less sensitive to the inhibitors, showing Because inhibition or knockdown of cathepsin B had no effects on ϳ50% reduction even at a maximum concentration of 100 ␮M TNF-␣ mRNA levels (Fig. 2B), we examined whether cathepsin B ␮ ␣

CA-Me (Fig. 3B, right). CA-Me at the concentration of 100 M was involved in translation or secretion of TNF- . To examine by guest on September 27, 2021 had no further suppressive effects on TNF-␣ production in CtsbϪ/Ϫ whether cathepsin B is involved in the translation of TNF-␣ in murine macrophages (Fig. 3B, right), confirming that the TNF-␣ BMDIM, pro-TNF (26-kDa) protein was immunoprecipitated after suppressive effect of CA-Me was specifically due to inhibition of radiolabeling cells were pulsed [35S]methionine for 1 h and chased cathepsin B activity at this concentration. Cathepsin B and cathep- for the next 90 min in the presence or absence of CA-Me. No sin L inhibitor, zFF-fmk had similar inhibitory effects on TNF-␣ significant difference in the levels of newly synthesized pro-TNF

FIGURE 3. TNF-␣ production in human monocytes is more sensitive to the CA-Me than murine macrophages. A, Human monocytic cell line THP-1 cells were transfected with siRNA against cathepsin B (siCTSB) or scrambled siRNAs (CNT) for 48 h. Cells were then plated on a new 24-well plate and stimulated with LPS (1 ␮g/ml) for 5 h. Cell culture medium was collected for TNF-␣ assay and cells were used for Western blot analysis for cathepsin B and p38 MAPK for loading control. B, THP-1 cells pretreated (left) with various concentrations of CA-Me for 30 min were stimulated with LPS (1 ␮g/ml) for 5 h. Similarly, bone marrow-derived macrophages from wild-type C57BL/6 (Ctsbϩ/ϩ) and CtsbϪ/Ϫ mice were pretreated (right) with CA-Me (100 ␮M), cathepsin B and cathepsin L inhibitor zFF-fmk (FF; 50 ␮M), cathepsin K inhibitor (CtsKi; 50 ␮M), cathepsin G inhibitor (CtsGi; 50 ␮M), and calpain inhibitor I (50 ␮M) for 30 min and then stimulated with LPS (1 ␮g/ml) for 3 h. C, Freshly isolated human PBMC were treated with CA-Me (50 ␮M) or brefeldin A (BFA) for 30 min and then stimulated with LPS (1 ␮g/ml) for 5 h. B and C, TNF-␣ in culture medium was measured by bioassay. Data are expressed as mean Ϯ SD of over three experiments. 694 CATHEPSIN B IN TNF-␣-CONTAINING VESICLE TRAFFICKING

FIGURE 4. CTSB inhibition induced intracellular accumulation of pro-TNF. A and B, BMDIM pretreated with or without CA-Me (50 ␮M) for 45 min were treated with LPS (1 ␮g/ml) for 90 min and then metabolically labeled with [35S]methionine for 60 min. Cell lysates and culture medium were collected at times 0, 60, and 90 min after [35S]methionine labeling. Labeled TNF-␣ in cell lysates (A) and culture medium (B) were immunoprecipitated and visualized by autoradiography. The intensity of the bands was analyzed using the NIH Image program. Data are expressed as mean Ϯ SD (n ϭ 2 experiments). C, BMDIM pretreated with or without CA-Me (50 ␮M) were treated with LPS (1 ␮g/ml) for the times indicated in the experiment, and TNF-␣ was immunoprecipitated and blotted with TNF-␣ Ab. Downloaded from was detected between CA-Me-treated and nontreated cells (Fig. much less GFP association with the plasma membrane, whereas 4A, second and sixth lanes). However, labeled pro-TNF was de- Cre-expressing CTSBmut-Cre clones showed GFP associated with tected for signiﬁcantly longer time periods in CA-Me-treated than the plasma membrane (Fig. 5C). Similar results were also obtained in nontreated cells (Fig. 4A, bottom). At the same time, the cleaved soluble form of radiolabeled 17-kDa TNF-␣ was detected at sig-

nificantly higher levels in nontreated than in CA-Me-treated cell http://www.jimmunol.org/ culture medium (Fig. 4B). Consistently, Western blot analysis for the overall amounts of intracellular pro-TNF indicated that CA- Me-treated cells contained higher levels after 6 h (Fig. 4C). A higher level of intracellular TNF-␣ in CA-Me-treated cells was confirmed by ELISA of cell lysates (data not shown). These results suggest that cathepsin B is involved in posttranslational TNF-␣ production steps, either in TNF-␣-containing vesicle trafficking to the plasma membrane or in the release of TNF-␣ from the plasma membrane into culture medium. by guest on September 27, 2021

TNF-␣-containing cargo vesicles fail to reach the plasma membrane in the absence of cathepsin B To visually examine where the defect in TNF-␣ production orig- inates in the absence of cathepsin B, TNF-␣ was immunostained in cells treated with LPS with or without CA-Me. LPS alone slightly increased TNF-␣ staining inside cells in a distinct puncta (Fig. 5A). However, TNF-␣ staining was further enhanced inside cells treated with LPS together with CA-Me. The TNF-␣-specific staining was highly localized at a focal point close to the plasma membrane but was absent from the plasma membrane. These data suggest that cathepsin B is involved in trafficking TNF-␣-containing vesicle, rather than cleavage of pro-TNF on the surface of cell membrane. However, it is still possible that the high intensity of TNF-␣ immunofluorescence was due to an accumulation of uncleaved pro- TNF recycled into cytosol because uncleaved pro-TNF is endocy- tosed and appears in intracellular vesicles (27). To address this question, we ectopically expressed N-terminal GFP-conjugated FIGURE 5. Cathepsin B is involved in trafficking of TNF-␣-containing pro-TNF in BMDIM in the presence or absence of CA-Me. GFP vesicles to the plasma membrane. A, Intracellular immunostaining of en- conjugation to the N terminus of pro-TNF allows GFP to remain dogenous TNF-␣. BMDIM grown on coverslips were treated with LPS in within the plasma membrane after cleavage of the C-terminal por- the absence or presence of CA-Me (50 ␮M) for 12 h or with scrambled tion of pro-TNF by TNF-␣-converting enzyme. Therefore, the siRNA (control) cells (CNT). Cells were then fixed, immunostained with presence of GFP in the plasma membrane indicates that pro-TNF TNF-␣ Ab, and visualized using an Axioscope 2 microscope. TNF-␣-con- has reached the plasma membrane. As expected, GFP was detected taining vesicles were strongly detected in a distinct puncta at a focal point close to plasma membrane only in cells treated with CA-Me. B, N-terminal within the cytosol as distinct puncta and highly detected on the GFP-conjugated pro-TNF was transiently transfected in the absence or plasma membrane after8hoftransient transfection of GFP-con- presence of CA-Me (50 ␮M) for 8 or 16 h in BMDIM and visualized using jugated pro-TNF (Fig. 5B). However, in cells treated with CA-Me, a confocal microscope. C, Similarly, CTSBmut or CTSBmut-Cre were tran- GFP was only detected in cytosol in distinct puncta and absent in siently transfected with N-terminal-conjugated pro-TNF. After 8 h, cells mut the plasma membrane even 16 h posttransfection. Most CTSB were stained with Hoechst 33258 dye for nuclear staining, and GFP-con- cells also showed an accumulation of GFP in cytosol in puncta but jugated TNF-␣ and nucleus were visualized using a confocal microscope. The Journal of Immunology 695

FIGURE 6. Intracellular cathepsin B activity is required for trafﬁcking of TNF-␣-containing vesicles to the plasma membrane. A, BMDIM pretreated with various concentrations of CA074 or CA-Me for 30 min were stimulated by LPS (100 ng/ml) for 5 h. TNF-␣ presented in cell culture medium were analyzed using bioassay. Data are expressed as mean Ϯ SD (n ϭ 3 experiments). B, BMDIM were pretreated with CA074 or CA-Me (50 or 100 ␮M) for 30 min and treated with LPS (100 ng/ml) for 18 h. The presence of intracellular TNF-␣ was measured using FACS analysis. Dat shown are representative results of similar observations in three independent experiments.

in Ctsbϩ/ϩ and CtsbϪ/Ϫ bone marrow-derived macrophages, re- monocytic cell line THP-1 cells in responses to endocytosis (32) or spectively (data not shown). These results suggest that cells lack- inﬂammatory stimuli such as LPS and IFN-␥ (33, 34). We show in ing cathepsin B have a defect in transporting GFP-conjugated pro- this study that cathepsin B is involved in the posttranslational reg-

TNF to the plasma membrane. ulation of TNF-␣, likely in the trafficking of TNF-␣ containing Downloaded from vesicles to the plasma membrane. Based on the results showing Intracellular cathepsin B activity is required for TNF-␣ much higher sensitivity to the membrane permeable version of secretion CA-Me than to the less-permeable homolog CA074 in TNF-␣ se- Cathepsin B can be secreted to pericellular compartments and af- cretion (Fig. 6), intracellular rather than pericellular cathepsin B fect TNF-␣ release from the plasma membrane. To examine activity is likely involved in TNF-␣ secretion. In CtsbϪ/Ϫ-orCA- whether pericellular cathepsin B activity was involved in the pro- Me-treated macrophages, the ectopic expression of GFP-conju- http://www.jimmunol.org/ cess of TNF-␣ release, we compared the effects of two cathepsin gated pro-TNF showed a defect in reaching cell surface, further B inhibitor homologs, membrane permeable CA-Me with the less substantiating the notion that cathepsin B is not involved in the permeable form of the inhibitor CA074. CA-Me is a proinhibitor, cleavage of pro-TNF or TNF-␣-converting enzyme at the cell sur- which is converted to CA074 after internalization (28). The effec- face (Fig. 5). However, this study could not delineate whether the ␣ tiveness of CA-Me in suppressing TNF- production was much site of cathepsin B action was in the cytoplasm, lysosomes, or greater than that of CA074 (Fig. 6A). We further analyzed the another intracellular compartment. Cathepsin B was shown to be ␣ accumulation of TNF- in the cytosol using flow cytometry. In released into the cytosol in certain tumor cells (21) and cells agreement with fluorescence microscopy and Western blot data treated with different stimuli such as TNF-␣ or the Streptomyces ␣ by guest on September 27, 2021 (Figs. 4 and 5), newly synthesized TNF- was quickly released toxin nigericin (19, 20) and in cells infected with parvovirus H-1 from cells, resulting in only slight intracellular accumulation of (35). Although total cathepsin B activity was not changed by LPS TNF-␣ in either LPS alone or LPS plus CA074-treated (50 ␮M) within5hofthetreatment, we detected a small amount of cathep- cells. In contrast, CA-Me enhanced intracellular TNF-␣ accumu- sin B released into the cytosol as early as 30 min of LPS treat- lation over an 18-h period (Fig. 6B), suggesting that intracellular ments, based on Western blot analysis (data not shown). However, cathepsin B activity in the cytosol, rather than in pericellular space, further definitive studies are required to conclude whether cytoso- is likely involved in TNF-␣ secretion. lic cathepsin B is required for the fusion of TNF-␣-containing Discussion vesicles to the plasma membrane. Recent studies have elucidated that the newly synthesized pro- To identify molecules that regulate TNF-␣ synthesis and secretion, TNF is initially accumulated in the Golgi complex (36). Pro-TNF- we used a retrovirus-mediated random mutagenesis in macro- containing vesicles are then transported from the trans-Golgi net- phages. Each mutant clone was analyzed for TNF-␣ production in work to the recycling endosome and subsequently to the cell response to LPS treatment. This procedure identified that cathepsin surface through two distinct membrane fusion processes (7, 37, B was required for optimal production of TNF-␣ in response to a TLR4 ligand LPS (Fig. 1). We further confirmed that cathepsin B 38). The first fusion process is mediated by Q-SNAREs, compris- is also required for TNF-␣ production in response to a TLR2 li- ing syntaxin 6, syntaxin 7, and Vit1b (vesicle transport through gand lipoteichoic acid and TLR9 ligand CpG (Fig. 2C) at post- interaction with t-SNAREs homolog 1b), of the Golgi-complex ␣ translational levels (Fig. 2B) and in both mouse and human mac- TNF- carrier vesicle and the R-SNARE VAMP3 (vesicle-asso- rophages or monocytes (Fig. 3). ciated membrane protein 3) of the recycling endosome. The second Cathepsin B is primarily known as a lysosomal cysteine pro- fusion process is through interaction between the VAMP3 of the tease involved in the degradation of cellular proteins, bone resorp- recycling endosome and the Q-SNARE complex of the plasma tion, trypsinogen activation, and cell death (29, 30). Recent studies membrane, comprising synthaxin 4 and SNAP-23. Activation of further showed that cathepsin B is released to other sites of cells macrophages by LPS induces expression of Q-SNARE compo- and involved in various cellular functions. Cathepsin B is secreted nents synthaxin 4 and SNAP-23 to accommodate the increased to pericellular environments where it enhances tumor cell inva- trafficking during TNF-␣ secretion (38). Cathepsins such as ca- siveness (16), myoblast differentiation (17), and survival of cyto- thepsin L (39) or cathepsin B (40, 41) can be localized in the toxic lymphocytes after degranulation (18). In certain conditions, nucleus and regulate transcription factors. We did not detect cathepsin B is released to cytosol and triggers arachidonic acid differences in the levels of synthaxin 4 and SNAP-23 in CA- release (19), caspase-1 or caspase-11 activation (20, 31), and tu- Me-treated or CtsbϪ/Ϫ macrophages (data not shown), suggest- mor cell invasion (21). A rise in intracellular cathepsin B activity ing that cathepsin B is not inhibiting fusion of TNF-␣-contain- has been observed long ago in peritoneal macrophages or human ing vesicles to the plasma membrane by down-regulating these 696 CATHEPSIN B IN TNF-␣-CONTAINING VESICLE TRAFFICKING

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